U.S. patent application number 10/520016 was filed with the patent office on 2006-06-29 for human igm antibody lysing activated lymphocytes under mediation by homologous complement.
Invention is credited to Hidechika Okada, Noriko Okada.
Application Number | 20060140964 10/520016 |
Document ID | / |
Family ID | 30002401 |
Filed Date | 2006-06-29 |
United States Patent
Application |
20060140964 |
Kind Code |
A1 |
Okada; Hidechika ; et
al. |
June 29, 2006 |
Human igm antibody lysing activated lymphocytes under mediation by
homologous complement
Abstract
A human IgM monoclonal antibody responding to HIV-infected cells
too, which is characterized by lysing activated human lymphocytes,
etc. under the mediation by a homologous human complement, is
obtained. Using the thus obtained monoclonal antibody, it is
intended to provide an immune reaction controlling remedy, etc.
containing the human IgM monoclonal antibody which responds
specifically to activated lymphocytes and induces cell lysis under
the mediation by a homologous complement. Using the human IgM
monoclonal antibody responding to activated human lymphocytes, it
is also intended to provide an HIV remedy, etc. characterized by
lysing and eliminating activated lymphocytes to thereby treat
transplantation rejection and autoimmune diseases caused by an
over-response of T lymphocytes as well as HIV infection
Inventors: |
Okada; Hidechika; (Aichi,
JP) ; Okada; Noriko; (Aichi, JP) |
Correspondence
Address: |
ADAMS EVANS P.A.
2180 TWO WACHOVIA CENTER
CHARLOTTE
NC
28282
US
|
Family ID: |
30002401 |
Appl. No.: |
10/520016 |
Filed: |
June 30, 2003 |
PCT Filed: |
June 30, 2003 |
PCT NO: |
PCT/JP03/08306 |
371 Date: |
October 20, 2005 |
Current U.S.
Class: |
424/160.1 ;
530/388.3 |
Current CPC
Class: |
A61P 37/06 20180101;
A61P 31/18 20180101; C07K 16/28 20130101; A61K 2039/505 20130101;
C07K 2317/21 20130101; C07K 2317/56 20130101; Y10S 530/809
20130101 |
Class at
Publication: |
424/160.1 ;
530/388.3 |
International
Class: |
A61K 39/42 20060101
A61K039/42; C07K 16/10 20060101 C07K016/10 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 1, 2002 |
JP |
2002-227952 |
Mar 18, 2003 |
JP |
2003-74312 |
Claims
1. A human IgM monoclonal antibody reactive to activated human
lymphocytes and HIV-infected cells for lysing cells under mediation
by homologous human complements.
2. An immunosuppressant and HIV remedy for treating transplantation
rejection responses and autoimmune diseases caused by excessive
reaction of T-lymphocytes, whereby abnormally activated lymphocytes
are eliminated by cytolysis using a human IgM monoclonal antibody
reactive to activated human lymphocytes.
3. The human IgM monoclonal antibody according to claim 1 or 2,
wherein the human IgM monoclonal antibody reactive to the activated
human lymphocytes and HIV-infected cells is 9F11 antibody
comprising a base sequence of an H-chain variable region
represented by sequence number 1.
4. The human IgM monoclonal antibody according to any one of claims
1 to 3, wherein the human IgM monoclonal antibody reactive to the
activated human lymphocytes and HIV-infected cells is 9F11 antibody
comprising a base sequence of a L-chain variable region represented
by sequence number 2.
5. A cell strain with an accession No. of FERM PB-8379 that
produces the human IgM monoclonal antibody reactive to the
activated human lymphocytes and HIV-infected cells that lyses the
cells under mediation by a homologous human complement.
6. The monoclonal antibody according to any one of claims 1 to 4
produced by the cell strain with an accession No. FERM BP-8379.
Description
TECHNICAL FIELD
[0001] The invention relates to a human IgM monoclonal antibody
reactive to HIV-infected cells that lyses activated lymphocytes by
reacting with a differentiated antigen of the activated lymphocytes
under medication by homologous human complements, and a remedy for
autoimmune diseases containing the monoclonal antibody.
BACKGROUND ART
[0002] Various immunosuppressants such as cyclosporin and FK506
have been developed for controlling biological immunoreactions in
collagen diseases, autoimmune diseases and rejection for organ
transplantation. However, since such immunosuppressants are
reactive to cells other than immunocompetent cells, side effects of
these agent should be taken into consideration.
[0003] Various methods have been investigated for using antibodies
that specifically react with target cells. For example, the target
cell with which the antibody has reacted is expected to be lysed by
reacting with a complement. While there are species-specific
complement control membrane factors (such as DAF, decay
accelerating factor; MCP, membrane cofactor protein; and HRF20, 20
kDa homologous restriction factor) on human cell membranes, they
can induce no cytolysis reaction via complement reactions for
preventing reactions among homologous human complements.
[0004] On the other hand, it was found that IgM antibodies in human
serum that react with the HIV-infected cells are able to yield the
cytolysis reaction of the HIV-infected cells via the human
complement by overcoming the complement control membrane factors.
It was revealed that the IgM antibody can exhibit such action as
described above against gangliosides such as GM2 and Gg4 whose
expression is enhanced by HIV-infection (Japanese Patent
Application Laid-Open No. 9-227409).
[0005] L55 has been reported as the human IgM monoclonal antibody
against GM2 of the gangliosides, wherein L55 is produced by
immortalizing human B lymphoblast strain with EB virus. The
HIV-infected cells after treating with this human IgM monoclonal
antibody have been found to yield cytolysis via a reaction with the
human complement.
DISCLOSURE OF THE INVENTION
[0006] An object of the invention is to provides a remedy for
controlling immunological response containing a human IgM
monoclonal antibody that specifically reacts with activated
lymphocytes to induce cytolysis under mediation by homologous
complements.
[0007] As a result of intensive studies for solving the problems
above, the invention provides a human IgM monoclonal antibody
reactive to HIV-infected cells that lyse activated human
lymphocytes under mediation by homologous human complements.
[0008] The invention for solving the problems above provides an HIV
remedy that cures transplantation rejection response and autoimmune
diseases caused by excessive response of T-lymphocytes as well as
HIV infection diseases by eliminating activated lymphocytes by
cytolyses using a human IgM monoclonal antibody that reacts with
HIV-infected cells and activated lymphocytes.
[0009] More specifically, the invention for solving the problems
above provides the human IgM monoclonal antibody according to any
one of first to second aspects, wherein the human IgM monoclonal
antibody that reacts with the activated lymphocytes and the
HIV-infected cells is 9F11 antibody having a base sequence of the
H-chain variable region represented by sequence number 1.
[0010] More specifically, the invention for solving the problems
above provides the human IgM monoclonal antibody according to any
one of first to third aspects, wherein the human IgM monoclonal
antibody that reacts with the activated lymphocytes and the
HIV-infected cells is 9F11 antibody having a base sequence of the
L-chain variable region represented by sequence number 2.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 shows specificity of 9F11 antibody.
[0012] The result of flow cytometory analysis shows that
HIV-infected cells are stained with 9F11 antibodies while
non-infected cells are not.
[0013] FIG. 2 shows specificity of 9F11 antibody to peripheral
blood lymphocytes.
[0014] The drawing shows that 9F11 antibody reacts with the
activated lymphocytes stimulated with PHA while it does not react
with normal peripheral blood lymphocytes (PBMC: peripheral blood
lymphocyte).
[0015] FIG. 3 shows a cell impairing reaction by 9F11 antibody
under mediation by the complement.
[0016] (A) Almost all cells are destroyed 4 hours after addition of
2 .mu.g/ml of 9F11 antibody and fresh human serum (including a
complement component) to HIV-1 infected cells MOLT-4/IIIB. No
effects have been observed at all when the serum is not added or on
non-infected cells MOLT-4.
[0017] (B) 9F11 antigen is induced by activating the peripheral
blood lymphocytes using PHA, and the cells are impaired by 9F11
antibody and the complement as in the HIV-1 infected cells (FHS:
fresh human serum (used as a complement source), PHA: human
lymphocyte activating agent, %.sup.51Cr release=mortality of cells,
PBMC: peripheral blood lymphocyte).
[0018] FIG. 4 schematically illustrates 9F11 .mu.-chain expression
plasmid construct.
BEST MODE FOR CARRYING OUT THE INVENTION
[0019] While the invention is described in detail with reference to
examples, the technical scope of the invention is by no means
restricted to these examples.
[0020] For solving the problems above, the inventors of the
invention immunized HIV-infected cells of a mouse (TC mouse:
trans-chromosome mouse; prepared by Kirin Brewery Co., Ltd.) into
which human immunoglobulin gene-containing chromosomes had been
introduced, and obtained a mouse that produces human antibodies
that specifically react with HIV-infected cells. Hybridoma was
prepared by a conventional method by fusing spleen cells of the
immunized mouse with a mouse myeloma cell strain. Clones producing
the monoclonal antibody that react with the HIV-infected cells and
lead the infected cells to cytolysis in the presence of a human
complement were selected from the hybridoma. The selected
hybridomas were named as 9F11 cell strain. A 9F11 antibody that is
a monoclonal antibody produced by 9F11 cell strain is a human IgM
monoclonal antibody comprising human .mu.-chain and human
.kappa.-chain. While 9F11 antibody was able to yield cytolysis by
reacting with the HIV-infected cells under mediation by the human
complement, the antibody also caused cytolysis for activated
lymphocytes of non-infected lymphocytes. Accordingly, the response
to the HIV-infected cells may be comprehended that the lymphocyte
is brought to a certain kind of activated state by HIV infection,
and the HIV-infected cells are lysed under mediation by the human
complement by expressing an antigen (9F11 antigen) against 9F11 as
a differentiated antigen. In other words, 9F11 antigen is a
differentiated antigen that is expressed by activation of the
lymphocyte, and 9F11 antibody that induces cytolysis under
mediation by the complement by reacting with the antigen
specifically leads the activated lymphocyte to cytolysis under
mediation by the complement. It was therefore made clear that a
remedy containing 9F11 antibody can be utilized in the treatment
for suppressing the activated lymphocyte. The invention has been
completed based on the discoveries described above. The cell strain
9F11 that produces 9F11 antibodies of the invention was deposited
with National Institute of Advanced Industrial Science and
Technology, International Patent Organism Depository (Chuo-6,
Higashi 1-1-1, Tsukuba City, Ibaraki Pref.), on May 8, 2003, with
an accession number of FERM BP-8379.
[0021] Table 1 shows the results of base sequence analysis of the
genes in the variable regions in .kappa.-chain and .mu.-chain,
respectively, encoding 9F11 antibody. The base sequence of the
constant region is approximately the same as the base sequence of
reported genes.
Table 1
Base Sequence of .mu.-Chain Variable Region
Base Sequence of .kappa.-Chain Variable Region
[0022] The composition of the invention, which is used for an
immunological reaction control agent containing the human IgM
monoclonal antibody that specifically reacts with the activated
lymphocytes and induces cytolysis under mediation by the homologous
complement, may be obtained by combining with physiologically
acceptable carriers. The physiologically acceptable carrier is
known in the art, and includes physiological buffered saline or
other aqueous solutions having a buffer action, or solvents such as
glycols, glycerol, oils (for example olive oil) or injectable
organic esters. The physiologically acceptable carrier may include
compounds that stabilize human IgM antibody or enhance absorption
thereof. Examples of the physiologically acceptable compounds
include sugars such as glucose, sucrose and dextran; antioxidants
such as ascorbic acid and glutathione; chelating agents; proteins
such as albumin; and other stabilizers and excipients. Various
immunosuppressants such as cyclosporin and FK506 as well as other
immunosuppressants may be added to the composition. Any
combinations of the physiologically acceptable carriers may be
selected depending on administration path and disease of
object.
EXAMPLE 1
Specificity of 9F11 Antibody
[0023] Test cells were suspended in a culture medium in a
concentration of 1.times.10.sup.6 cells/ml, and an equal volume of
a 9F11 antibody solution in a concentration of 10 .mu.g/ml was
added to the suspension followed by reacting for 30 minutes. The
test cells were washed, linked 9F11 was stained with
fluorescence-labeled antihuman IgM antibody, and the cells were
analyzed by flow cytometory. The results showed that 9F11 antigen
is expressed by HIV infection since, although MOLT-4 cells and CEM
cells as human cell strains were not stained, the cells infected
with HIV-1 IIIB strain and MN strain were strongly stained (FIG.
1). Since HIV infection induces activation of the lymphocytes,
staining of the activated lymphocytes was investigated after
cultivating for 3 days by adding phytohemagglutinin (PHA) to the
lymphocyte in the peripheral blood cells of a normal person. It was
made clear that 9F11 antigen is a differentiated antigen expressed
in the activated T-lymphocytes (FIG. 2), since the activated
T-lymphocytes stimulated with PHA was strongly stained, although no
staining was observed in the peripheral blood cells and peripheral
blood lymphocytes not stimulated.
EXAMPLE 2
Cell Impairing Reaction by 9F11 Antibody under Mediation by
Complement
[0024] Test cells were labeled with .sup.51Cr as a radioactive
isotope in advance, and 40 .mu.l of 9F11 solutions in various
concentrations and 20 .mu.l of human fresh serum (complement serum)
were added to the labeled test cells (suspended in a culture medium
at a concentration of 5.times.10.sup.5/ml) to allow the mixture to
react for 4 hours on a microtiter plate. After the reaction, the
cells were precipitated by centrifugation of the plate, and the
radioactivity of .sup.51Cr released in the supernatant by cytolysis
was measured as an index of the cytolysis reaction. The cells
expressing 9F11 antigen such as MOLT-4/IIIB (MOLT-4 cells infected
with HIV-1) and peripheral blood derived lymphoblasts activated
with PHA were lysed under mediation by the complement in the
presence of 2 .mu.g/ml of 9F11 . On the contrary, no cytolysis was
observed by using an inactivated serum after heating human serum at
56.degree. C. or C9 deficient human fresh serum. Since cytolysis
occurs by adding purified C9 to the C9 deficient human fresh serum,
it was concluded that the human complement reaction is essential
for the cytolysis reaction (FIG. 3).
EXAMPLE 3
Reconstruction of Antibody by Gene Engineering
[0025] An example of the method for reconstruction of 9F11 antibody
based on the base sequence of the variable region of 9F11 antibody
shown in TABLE 1 will be described below, wherein 9F11
antibody-producing cell strains were established using gene
engineering such as a shot-gun ligation method (Grundstrom, T. et
al., Nucleic Acid Res. 13, 3305-3316, 1985).
[0026] Amino acid sequences of the variable region of 9F11 antibody
were obtained by translating the base sequence in the table. There
are many base sequences encoding the amino acid sequences in the
variable region of 9F11 antibody as shown in Table 2, such as the
base sequence of the variable region of original 9F11 antibody as
well as those obtained by changing used codons. Base sequences
having certain kinds of restriction enzyme recognition fragment
were selected from the sequences for every length capable of
chemically synthesizing as oligonucleotides (Table 2).
Table 2: Examples of cDNA Encoding Equivalent Amino Acids in the
Amino Acid Sequences of 9F11 Antibody
[0027] Oligonucleotides were chemically synthesized based on the
base sequence divided for each restriction enzyme-recognition
fragment. After sequentially digesting the synthesized
oligonucleotide with a corresponding restriction enzyme, a full
length of a base sequence encoding the amino acid sequence of the
9F11 antibody variable region by ligation was obtained. cDNA
fragments of the 9F11 antibody variable regions of the H-chain and
L-chain obtained by the same method with each other (named as
rV.mu.9F11 and rV.kappa.9F11 , respectively) were integrated into
vectors having constant region gene sequences of H-chain and
L-chain of the human IgM antibody (C.mu. and C.kappa.,
respectively) by the same method as forming chimera antibodies to
obtain recombinant 9F11.mu.-chain and .kappa.-chain expression
plasmids (rV.mu.9F11-C.mu. and rV.kappa.9F11-C.kappa.,
respectively; FIG. 4).
EXAMPLE 4
Expression of Recombinant Antibody
[0028] Activities of the antibodies obtained using the plasmids
expressing the reconstructed 9F11 antibody genes were investigated
with a temporary expression system in COS7 cells (ATCC CRL 1651).
Genes were introduced using a mixture of two plasmids
(rV.mu.9F11-C.mu. and rV.kappa.9F11-C.kappa.) and an expression
plasmid (Cj) for J-chain of human IgM antibody using a
lipofectamine reagent according to the protocol by GIBCO Co.
Cultivation was continued for two days thereafter under a usual
culture condition, and the supernatant of the gene-introduced cell
culture was retrieved. Recombinant 9F11 antibodies secreted in the
culture supernatant were confirmed by subjecting the culture
supernatant to an assay system by the sandwich ELISA method using
antihuman .mu.-antibody and antihuman .kappa.-antibody. The
antibodies were confirmed to have specificity as described above by
FACS analysis of the culture supernatant using cells such as
U937/IIIB and MOLT-4/IIIB prepared by infection of U937 cells and
MOLT-4 with IIIB strain of HIV-1. Further, the activity of
recombinant 9F11 antibody was confirmed by a competitive inhibition
test by allowing fluorescence-labeled original 9F11 antigen and the
culture supernatant to simultaneously react with U937/IIIB or
MOLT-4/IIIB.
[0029] Accordingly, it was confirmed that the base sequences of the
.mu.-chain and .kappa.-chain of 9F11 antibody listed in Table 1 are
quite important regions responsible for antibody activity against
differentiated antigens of the activated lymphocytes expressed in
the HIV-infected cells.
[0030] It was also confirmed from these results that genes encoding
the base sequences of the .mu.-chain variable region and
.kappa.-chain variable region are quite crucial genes for preparing
the recombinant anti-HIV antibody and anti-activated
lymphocytes.
INDUSTRIAL APPLICABILITY
[0031] Since the human IgM monoclonal antibody of the invention
against the differentiated antigens expressed in the activated
lymphocytes displays a cytolysis function for the activated
lymphocytes under mediation by the complement, the antibody can be
used as a remedy for controlling abnormally activated lymphocytes
in the body. The invention also provides genes encoding the base
sequences of the .mu.-chain variable region and .kappa.-chain
variable regions that are quite crucial for preparing the
recombinant anti-activated lymphocytes antibody.
* * * * *